Crystallization is the process of formation of solid crystals precipitating from a solution. It is also an industrial process; in chemical engineering crystallization occurs in a crystallizer. Crystallization is therefore an aspect of precipitation, obtained through a variation of the solubility conditions of the solute in the solvent, as compared to precipitation due to chemical reaction. Crystallization happens when atoms or molecules are arranged in a fixed lattice.
The importance of crystallization is that it serves as the basis for X-ray crystallography, wherein a crystallized molecule is used to determine the molecule's three-dimensional structure via X-ray diffraction. The first step in determining the structure of a molecule using X-ray crystallography is to grow crystals of sufficient quality to diffract X-rays strongly. X-ray crystallographic studies are of considerable interest to pharmaceutical and biotechnological industries, towards drug design and chemical engineering. By crystallizing and studying the molecules, one can understand mechanisms by which all the molecules function in biological systems. On the other hand, this knowledge can be useful in the ultimate development of new drugs.
Appropriate conditions for the production of crystals that produce a high-quality diffraction pattern must be found for each molecule before structure determination. However, since crystallization is relatively unpredictable, researchers have to perform a huge number of crystallization trials under various conditions. Molecules can be prompted to form crystals when placed in the appropriate conditions. In order to crystallize a molecule it undergoes slow precipitation from an aqueous solution. As a result, individual molecules align themselves in a repeating series of “unit cells” by adopting a consistent orientation.
Crystallization is a complex process, involving multiple equilibria between different states of the crystallizing species. The three stages of crystallization common to all molecules are nucleation, crystal growth and cessation of growth. During nucleation enough molecules associate in three dimensions to form a thermodynamically stable aggregate, the so called critical nucleus. These nuclei provide surfaces suitable for crystal growth, which can occur by a couple of different mechanisms. Crystal growth ceases when the solution is sufficiently depleted of spare molecules. Both crystal nucleation and growth occur in supersaturated solutions where the concentration of the crystallizing species, exceeds its equilibrium solubility value. The supersaturation requirements for nucleation and crystal growth differ.
The requirements for a molecule to crystallize are graphically described by the phase diagram as shown in FIG. 1.
In order to bring a molecule to the crystallization state there are different strategies, in general guiding the system to a state of reduced solubility. This can be achieved by changing one or more of many different parameters. A number of different parameters have been identified as affecting the crystallization process. Table 1 summarizes known parameters affecting crystallization.
TABLE 1CRYSTALLIZATION PARAMETERSPHYSICAL FACTORSCHEMICAL FACTORSBIOCHEMICAL FACTORSTemperaturePrecipitant typeSample purityMethodologyPrecipitant concentrationMacromolecular impuritiesTimepH and BufferAggregationPressureIonic strengthPosttranslational modificationsGravity, convection,Reducing/oxidizingSample source and storagesedimentationenvironmentVibrations/soundSample concentrationProteolysisMagnetic fieldsMetal ionsChemical and sequencemodificationsElectric fieldsDetergentsSample symmetryDielectric propertiesSmall molecule impuritiesSample pIViscosityPolyionsSample historyEquilibration rateCrosslinkersLigands and co-factorsNucleantsHeavy metalsContamination and impuritiesVolumeReagent sourcePurification methodologyParticulate/amorphousReagent puritymaterialSurface of crystallizationReagent formulationdevice
A huge number of methods have been developed to investigate and influence prenucleation, nucleation and crystal growth. The nucleation of small molecules and macromolecules is governed by the same principles. In brief, in the supersaturation state clusters and aggregates of molecules are formed. If the dimensions reach a critical point (in other words if the radius of the clusters exceeds a certain size) then new molecules will accumulate more rapidly and the real nucleus will be generated. Different models have been proposed for the nucleation with the “Fluid Aggregate” model being the most appealing.
Crystal growth proceeds by direct addition and development of intact 3-D nuclei. FIG. 2 attempts to describe all the steps that drive the crystal growth process.
The crystal growth involves a number of interactions and bonds and is governed by complicated kinetics and thermodynamic laws.
Electric Fields and Crystallization
In solution, the molecules are surrounded by a hydration cage which hinders interactions between the molecules by acting as a dielectric and shielding the electrostatic attraction between adjacent molecules. Crystallization can only occur upon the removal of these hydration cages in supersaturated protein solutions. If this cage is removed too quickly by evaporation or an external field, however, the molecules will not reach their native conformation and the solution will form a gel instead. Existing methods such as vapor diffusion, seeding, microfluidics, electric fields, and magnetic fields hence aim to accelerate this slow diffusion and increase protein saturation in the solution to enhance crystal nucleation and growth. Z. Hammadi, Prog Biophys Mol Biol. 2009 November; 101(1-3):38-44 has reported that a localized, internal, electric field and direct current in agarose gel on the nucleation and growth of 2 biomolecules (one of which being Lysozyme) can control the whole nucleation process and can be used as a screening methodology. It is also proposed as a seeding technique for the crystal growth. The localized electric field was created by a nanometer size electrode tip, capable of generating an intense electric field.
H. Koizumi and collaborators, Langmuir 2011, 27:8333-8, have reported that an electric field can increase the nucleation rate of Hen Egg white lysozyme. In more details they have described a technique for the crystallization of proteins under the application of an AC electric field generated and controlled by an Electric Double Layer (EDL) which was formed at the inner surface of a protein solution drop immersed in low-density paraffin oil. Additionally, there are a large number of researchers who have tried to influence the crystallization process by electrostatic or current-injection fields. Taleb et al, 3. Cryst. Growth 1999, 200:575-82; Taleb et al, 3. Cryst. Growth 2001, 232: 250-55; Penkova et al, 3. Cryst. Growth 2005, 275:e1527-32 and many more have reported that a DC voltage, when applied to a protein solution (the protein being Lysozyme), was found to decrease the nucleation rate for all cases. On the other hand Hou and Chang, Appl. Phys. Lett. 2008, 92:223902, have reported the rapid increase in the crystal size of lysozymes by applying an AC current-injection field.
Methods of Crystallization
The conventional method of vapor diffusion, with either hanging or sitting drop, is usually adopted for the crystallization of the molecules. In this technique, a drop containing an amount of the molecule, stabilizing buffers, precipitants, and crystallization aids is allowed to equilibrate in a closed system with a much larger reservoir buffer. The reservoir usually contains the same chemicals, minus the molecule, but at an overall higher concentration, so that water preferentially evaporates from the drop; thus, it produces a gradual increase in molecule concentration and, under the right conditions, sometimes leads to the formation of crystals. Manual and automatic high-throughput approaches are used (in the lab or by commercial companies respectively).
A. Manual Crystallization Trials
Special hanging and sitting drop plates are used and a set of crystallization conditions is tested.    a. The wells of special 24-96 well crystallization plates are filled with 1 ml of various crystallization reservoir buffers (kits available with sets of 48 different crystallization buffers)    b. Drops (1-3 μl) of a concentrated molecule (2-50 mg/ml) are mixed with an equivalent volume of reservoir buffer and placed either on pre-siliconized cover slips (for hanging drop) or on special bridges (for sitting drop) so that the mix stays over the reservoir buffer (FIG. 3).    c. Plates are stored at different temperatures (4, 16, and 25° C.) and the wells are regularly inspected for crystal formation.    d. Formed crystals are tested to determine whether they are indeed formed of the molecule we wish (e.g. proteins are tested by Izit Crystal Dye, Hampton Research).B. Crystallization Trials by Robotics
Crystallization robots have been developed to automate and speed up the experimental process of crystal growth. Crystal screening is also attempted using crystallisation robots provided as service by companies described as “structural genomics” companies (e.g. Structural GenomiX and Syrrx) or in-house. Crystallisation robots enable many crystallisations to be performed and minimise the amount of protein used. In fact, this system saves protein (50 nl instead of 1 μl sample per drop) thus allowing more screenings to be performed, saves time, and it is more reproducible. It is mainly used for the initial crystallization trials.    a. Sitting drop crystallization trials (50 nl protein+50 nl mother liquor) in 96-well plates. Each plate is imaged by conventional photonic microscope.    b. After identification of the proper crystallization conditions, trials are repeated by the manual approach and are further refined by slight modifications for the improvement of the crystal quality.
A list of relevant literature concerning crystallization is provided here:    1) A. McPherson, Crystallization of biological macromolecules (Spring Harbor Laboratory Press, New York, 1999).    2) P. C. Weber, Overview of protein crystallization methods, Methods in enzymology A 276 (1997)13.    3) A. McPherson, Review: current approaches to macromolecular crystallization, Eur. J. Biochem. 189 (1990) 1.    4) Poulas K, Eliopoulos E, Vatzaki E, Navaza J, Kontou M, Oikonomakos N, Acharya K R, and Tzartos S J (2001). Crystal structure of Fab198, an efficient protector of acetylcholine receptor against myasthenogenic antibodies. Eur. J. Biochem. 268: 1-10.